The Leishmania parasite causes human disease with clinical symptoms ranging from-self healing cutaneous lesions to a fatal visceral infection. Additionally, in endemic areas, people infected with HIV are especially prone to Leishmania infection and latent infections can reactivate upon acquisition of HIV. The lack of understanding of cell biology and pathogenic mechanisms of this parasite makes the task of controlling this grave, worldwide health risk difficult. Closer to home, it is particularly of concern to U.S. military personnel, their families and other travellers visiting or living in the endemic areas. To find novel methods for control of this pathogen, we have initiated study to understand the mechanism of parasite differentiation from the avirulent (promastigote) to virulent (amastigote) form. This transformation is controlled by the genetic changes which take place in the parasite. We have developed a culture system which mimics the differentiation of Leishmania parasite from promastigotes to amastigotes. Using this culture system we have identified and cloned differentially expressed genes. The genomic clones for two such genes were isolated and complete sequence analysis revealed one with an open reading frame (ORF) of 482 amino acids predominantly expressed in promastigotes and a second with a 902 amino acid ORF predominantly expressed in amastigotes. Database searches indicated that no homologues of these proteins have been cloned from other organisms. The genetic changes that regulate differentiation should preceed the morphological changes, therefore we are attempting to study the expression of differentially expressed genes immediately after the parasite differentiation is initiated, and thus described as immediate early genes. RNA samples were collected before and at time intervals (1, 2, 4, 8, 16, 24, and 48 hrs) after induction of differentiation from promastigotes and amastigotes. These RNAs were hybridized with four differentially regulated genes including the two that were mentioned above. The results suggested that genes which were turned off over the course of differentiation did so in as little as 1 hour; while the genes that are up-regulated, gradually increased to their peak by 16 hours. Thus the immediate early genes could be used as targets for blocking the process of differentation in Leishmania. Further, the differential kinetics of expression of these genes indicates that these RNA samples can be used to generate probes to screen libraries for other novel genes which are expressed immediately after the initiation of differentiation. We have initiated collaboration to screen a L. major microarray for immediate early genes and have begun development of a L. donovani microarray. These novel genes may provide drug targests to block parasite differentiation or be useful to create attenuated parasite vaccines.